The present invention relates to a DNA fragment encoding a protein involved in fatty aldehyde decarbonylase activity, which protein is involved in the biosynthesis of alkanes. The invention also relates to a recombinant DNA containing the above fragment and to a method for obtaining transformed host cells using said recombinant DNA. The present invention further relates to transformed bacterial cells and to a method for producing a protein having fatty aldehyde decarbonylase activity using said transformed cells. Further, the invention relates to a method for obtaining a transformed plant showing an altered epicuticular wax composition and to a transformed plant, cell, fruit, seed of progeny derivable from said plant.
Plants are covered by an epicuticular wax (EW) layer composed of long chain lipids (C20-C40) consisting mostly of fatty acids, alcohols, esters and alkanes (Kolattukudy, 1976). There is a large variety of specific wax components which differ dramatically between plant species and thereby contribute towards the typical characteristics of the surface of individual plant species (Kolattukudy, 1975). This unique EW layer mediates the specific interactions of each plant species with its environment and is the first line of defense against abiotic and biotic stress, like drought/frost, pathogens and insects.
The primary function of the EW layer is to reduce water loss through the epidermis (Hall and Jones, 1961), a feature which contributes to drought tolerance. In addition individual lipid components or EW extracts and the physical structure of plant surface wax can influence insect behaviour which may lead to resistance of the plant to specific insects (Thompson, 1963, Stxc3xa4dler, 1986, Eigenbrode and Espelie, 1995). Additionally the EW layer has a major function in the interaction of plants with plant pathogenic fungi (Podila et al., 1993). EW is also used by humans. The wax palm produces wax that is used for commercial purposes such as carnauba wax for polishing while similar types of waxes are collected from various other plant species.
The EW biosynthesis pathway has been suggested to be based on an elongation-reduction-decarboxylation mechanism which generates long chain fatty acids, aldehydes and alkanes (Bianchi et al., 1985; Lemieux et al., 1994; von Wettstein-Knowles, 1979; von Wettstein-Knowles, 1994). Thus, wax synthesis is determined by various biochemical steps suggesting that many genes are involved. This is in line with the large amount of mutant loci that have been observed for several species such as maize, barley, Brassica spp. and Arabidopsis thaliana (Baker, 1974; von Wettstein-Knowles, 1979; Bianchi et al., 1985; Kolattukudy, 1980; Koornneef et al., 1989; McNevin et al., 1993; Lemieux et al., 1994). Most of the mutants show a bright green wax-free phenotype compared to the glaucous appearance of wild type plants due to reduced wax production. Mutants are termed glossy (gl) for maize and Brassica spp. and eceriferum (cer) for barley and Arabidopsis. The natural gl and cer mutants, selected on visual and therefore wax structure basis, are dramatic mutants which have changes in lipid composition as well as crystal structure. In Brassica species, the gl mutants have been shown to be resistant to specific insects (Stoner, 1992).
The modification of the biosynthesis pathway of EW in plants by genetic engineering approaches will enable the modification of EW and consequently the interaction between the plant and its environment. Accordingly changes in the EW components will provide novel systems to engineer plants resistant to (a)biotic stress. In addition the modification of the biosynthesis route also opens the possibility to provide plants or bacteria with new waxes with industrial application including pharmaceuticals, cosmetics, detergents, plastics and lubricants.
In general the biochemical steps in Arabidopsis consist of a series of elongation reactions up to C30 chain fatty acids which can be either reduced to alcohols or reduced and decarbonylated to alkanes. Further analysis of the enzymes responsible for the formation of EW in plants has been hampered by the lack of purified enzymes involved in the EW biosynthesis. It is desirable therefore, for further study of the EW biosynthesis pathway to devise a strategy whereby these enzymes can be identified. The protein products encoded by CER genes are probably membrane bound and difficult to isolate biochemically. Therefore, in order to accomplish such isolation and to be able to modify the wax biosynthesis route, the object of this invention is to isolate the genes which encode the enzymes involved in EW biosynthesis.
To this end we analysed the cer mutants of Arabidopsis thaliana at the molecular level. Arabidopsis thaliana is particularly suited for the isolation of the genes involved in EW biosynthesis, especially as 22 loci affecting EW are already known (Koornneef et al., 1989; McNevin et al., 1993). Biochemical compositional analysis of these mutants has enabled functions to be attributed to many of the loci. By the application of the transposon tagging technology we were able to isolate a gene responsible for the synthesis of a protein which is involved in the decarbonylation of fatty aldehydes to alkanes. In conformity with the previous indication of the loci, the isolated gene is named CER1 gene and the corresponding protein is named CER1 protein (Aarts et al., 1993; Aarts et al., 1995).
Relevant Literature
A review on the biosynthesis and genetics of waxes in plants has been published by von Wettstein-Knowles (1995) in: Waxes, The Oily Press, Dundee, Scotland; Ed. J. R. Hamilton. A paper on the analysis of leaf epicuticular waxes of the Eceriferum mutants in Arabidopsis has been published by M. A. Jenks et al., 1995 in Plant Physiology, Volume 108, pages 369-377.
Eigenbrode and Espelie published a review on the effects of plant epicuticular lipids on insect herbivores in Annual Review of Entomology 1995, Volume 40 pages 171-194.
Definitions
Gene or sense gene: a nucleotide sequence that can be expressed as RNA molecule and/or polypeptide.
Promoter: a nucleotide sequence which directs the expression of a (sense-) gene or antisense gene, or nucleotide sequences derived thereof.
Antisense gene: a nucleotide sequence having a homology of more than 50%, preferably more than 80% with the target gene as defined herein, and which is linked to a promoter in 3xe2x80x2 to 5xe2x80x2 orientation with respect to the target gene and can be expressed as an RNA molecule.
Inhibitor gene: a (sense-) gene or antisense gene, expression of which leads to prevention or inhibition of the expression of a target gene as defined herein.
Target gene: a gene which activity is to be inhibited by proper expression of an inhibitor gene as defined herein.
The present invention describes the isolation of the CER1 gene involved in EW synthesis of Arabidopsis thaliana. The EW of cer1 mutants has been analysed previously and found to be especially rich in aldehydes while lacking alkanes. This shows that the CER1 protein encoded by the provided CER1 gene is involved in the conversion of aldehydes to alkanes.
The present inventors have isolated the CER1 gene from a genomic Arabidopsis thaliana DNA library in phage lambda which encodes a protein indicated as CER1 which is involved in EW synthesis, said gene having the nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1). FIG. 1 shows the partial nucleotide sequence of the CER 1 gene, it being assumed that the complete nucleotide sequence will comprise about 6000 nucleotides. Further, the CER1 cDNA was isolated having the nucleotide sequence as shown in FIG. 2. (SEQ ID NO:2). The amino acid sequence of the CER1 protein deduced from said cDNA is shown in FIG. 4 (SEQ ID NO:5).
Accordingly, the present invention provides a DNA fragment encoding a protein comprising the amino acid sequence depicted in FIG. 4 (SEQ ID NO:5), or a protein substantially homologous therewith and involved in fatty aldehyde decarbonylase activity. Further the invention provides a DNA fragment comprising a nucleotide sequence depicted in FIG. 2 (SEQ ID NO:2), or a homologous nucleotide sequence. Said DNA fragment will hereinafter often be referred to as CER1 gene, it being understood that it is actually the CER1 cDNA. By xe2x80x9chomologous nucleotide sequencexe2x80x9d is intended any nucleic acid sequence that may be synthesized from the CER1 protein amino acid sequence, or alternatively identified in a different organism and isolated using CER1 protein nucleic acid sequences or antibodies prepared against the CER1 protein as probes. In this manner it can be seen that sequences that are isolated from other organisms using the CER1 sequences, either by nucleic acids or by antigenic methods, may similarly be used to isolate other proteins having the same activity. Such proteins are likewise considered as homologous.
Further, this invention provides the 5xe2x80x2 regulatory region or promoter of the CER1 gene isolated from the chromosomal DNA of Arabidopsis thaliana, said promoter having the nucleotide sequence as shown in FIG. 3 (SEQ ID NO:4) or a promoter of a homologous gene.
Further, the invention provides recombinant DNA molecules (recombinant nucleic acid sequences) comprising a DNA fragment as defined above. In particular, the present invention provides recombinant nucleic acid sequences which can be suitably used for obtaining host cells showing altered fatty aldehyde decarbonylase activity essentially comprising a gene capable to (over)express the CER1 gene in said host cell, encoding the CER1 protein, said gene having the sequence as shown in FIG. 2 (SEQ ID NO:2) or a homologous gene. In particular host cells which contain the preferred substrates of the CER1 protein such as cells of Brassica plants are considered. In another preferred embodiment according to the invention the promoter that is driving the expression of said gene comprises the promoter of the CER1 gene, said promoter having the sequence as shown in FIG. 3 (SEQ ID NO:4).
The present invention also provides recombinant nucleic acid sequences which can be suitably used for obtaining host cells showing an altered fatty aldehyde decarbonylase activity essentially comprising an inhibitor gene capable of inhibiting expression of a target gene in the said host cell. In the preferred embodiment of the invention the target gene is the CER1 gene, said gene having the sequence as shown in FIG. 1 (SEQ ID NO:1) or a homologous target gene.
Further the present invention provides recombinant nucleic acid sequences which can be suitably used for obtaining a host cell showing an altered alkane biosynthesis, essentially comprising an inhibitor gene capable of inhibiting expression of a target gene encoding the CER1 protein involved in alkane biosynthesis in the said host cell, said target gene having the sequence as shown in FIG. 1 (SEQ ID NO:1) or a homologous target gene.
In the preferred embodiment of the invention the target gene is the CER1 gene having the sequence as shown in FIG. 1 (SEQ ID NO:1). In yet another preferred embodiment of the invention the inhibitor gene is a (sense-)gene or antisense gene directed against said target gene. In yet another preferred embodiment according to the invention the promoter that is driving the expression of the inhibitor gene comprises the promoter of the CER1 gene, said promoter having the sequence as shown in FIG. 3 (SEQ ID NO:4).
The present invention also provides a method for obtaining a plant with altered EW composition comprising the steps of:
a. transferring any recombinant nucleic acid sequence according to the invention to a plant cell,
b. generating whole new plants from cells having incorporated said recombinant nucleic acid sequence, and
c. selecting a plant that has an altered EW composition.
Yet another embodiment of the invention is a recombinant genome, comprising incorporated therein a recombinant nucleic acid sequence according to the invention. In particular a plant genome or a bacterial genome is considered. Generally, the invention provides a method for obtaining a transformed host cell by transferring a recombinant DNA molecule as defined above, to the host cell. Said recombinant DNA molecule preferably comprises a DNA fragment encoding the CER1 protein or a homologous protein, operably linked with a DNA sequence capable of effecting (over)expression of said DNA fragment in the host cell. In particular bacterial host cells are considered. The thus transformed bacterial cell can be used for the production of the CER1 protein or a homologous protein. Accordingly, the invention provides a method for producing a CER1 protein or a homologous protein, comprising culturing the transformed bacterial cell in a suitable culture medium and isolating the protein.
The invention further encompasses transformed plants and cells, fruit, seed or progeny derivable from said plants through crossing or selfing.